The homeobox-containing genes Hoxa-1 and Hoxa-2 direct a major part of the embryonic development of the craniofacial and hindbrain regions. Null mutations in either gene result in severe craniofacial dysmorphology with associated neurological defects. Now that the mutation of all murine Hox genes has been cumulatively achieved in various laboratories, there remain three major areas of investigation to complete an understanding of how Hox genes direct embryonic development: 1) The identification of upstream regulators which initiate, maintain, and repress Hox gene expression in the correct temporal and spatial manner during embryogenesis; 2) The determination of the mechanism of homeoprotein binding specificity and subsequent gene activation; and 3) The identification of downstream targets (effectors) which mediate morphological diversity. This proposal focuses on the first of these three major areas. Owing to early deletion phenotypes, as well as overlapping roles from other Hox gene family members, mutant mice have not provided much information as to potential gene interactions or regulatory cascades. The identification and characterization of DNA regulatory sequences, followed by the characterization of potential protein regulators is currently the most successful means to directly dissect a mammalian regulatory pathway. After much initial experimentation with different systems, the Principal Investigator's primary approach has become focused on studies in animals (transgenic mice), since this is the only system which permits an accurate analysis of the complex spatial and temporal changes in gene expression which occur during embryonic development. Using this approach, they have identified two upstream regulators of Hoxa-1 gene expression. The first is the retinoic acid receptor alpha which is necessary for the initiation of Hoxa-1 gene expression in the caudal hindbrain and spinal cord. The second is the Hoxa-1 protein itself, which is involved in the maintenance of its own expression in rhombomere 4 via a positive autoregulatory interaction with the rhombomere 4-specific enhancer. In addition, they have recently identified a Hoxa-2 enhancer which functions equally well in flies and mice, indicating that the results obtained from these studies will impact on our understanding of Hox gene regulation in all animals.